Humans are losing the arms race against infectious bacteria as bacteria evolve resistance to broad-spectrum antibiotics. This is both a health problem and an environmental problem. The health problem is that we are losing the ability to effectively treat many bacterial infections. The environmental problem is that we are disrupting the microbial ecology around us by intensive use of antibiotics that kill bacteria indiscriminately. We need a new strategy that will target pathogens without affecting either commensal bacteria or the human host, and that will evolve continually as bacteria evolve to maintain control over infectious bacteria. The broad, long term objective of our application is to lay the foundations for such a new strategy. In this approach, instead of a broad spectrum antibiotic that kills many types of bacteria the bacteria would be infected by a synthetic virus, or virus-like particle, that brings into the bacterial cells antisense RNA. As the reader probably knows, one of the several uses of RNA in the cell is to regulate the expression of genes. We will emulate nature by designing RNA specifically to knock down critical genes in pathogenic bacteria only, and to have no effect on either beneficial bacteria or the human host. The virus-like particles will carry several different antisense RNA's, each of which separately will be sufficient to either kill or render nonvirulent the particular pathogenic bacterium that is being targeted. Bacteria will find it very difficult to evolve resistance to this approach for two fundamental reasons: 1) Because the antisense RNA's are designed to be specific to the pathogen's genome, they can be redesigned as the pathogen's genome changes, and 2) because the bacteria will be infected with multiple lethal RNA, there will not be a strong selection pressure favoring bacterial variants that can resist the effects of any one of the RNA's. The chances of a bacterium developing resistance to multiple lethal agents, when there is no selection advantage for developing resistance to any single agent, is small. Our research strategy rests on three areas of expertise: 1) bioinformatics to extract from the relevant genomic data all the antisense RNA sequences that could potentially knock down critical genes in the pathogenic bacteria, 2) nanoscience to induce the self-assembly of the virus-like particles from the constituent proteins and nucleic acids, and 3) microbial genetics and physiology to: a) add human knowledge to the computer outputs as a guide to prioritizing potential targets and b) to do the experiments on the pathogens in order to assess the effects of the antisense RNA. These three areas of expertise are embodied in the collaborating laboratories of Eric Jakobsson (bioinformatics), Jeff Brinker (nanoscience) and Stanley Maloy (microbiology). For this initial project we have chosen Salmonella as the target. Salmonella is an important pathogen whose genetics and mechanisms of virulence have been extensively studied, which will be a big advantage in interpreting experimental data. Also, Salmonella is acquiring resistance to several antibiotics. If we succeed in the laboratory with Salmonella, our strategy should be applicable to a wide variety of infectious diseases. PUBLIC HEALTH RELEVANCE: If the proposed research is successful, it will lay the foundations for transforming our approach to antimicrobial therapy. Broad spectrum antibiotics will be replaced with precisely targeted genetic controls aimed specifically at particular pathogens. The problem of antibiotic resistance will be overcome, as will the collateral damage done to beneficial microbiomes by antibiotics.